Project Summary Obesity, defined by the accumulation of excess body fat, is dramatically increasing worldwide and is driving an epidemic of type 2 diabetes. Adipose tissue and obesity are more complex than previously believed, and recent studies have shown adipose tissue is comprised of developmentally and functionally distinct subpopulations of adipocytes. To investigate this cellular heterogeneity, novel cellular and mouse models have been developed to identify and study subpopulations of white adipocytes both in vitro and in vivo. These studies have uncovered three distinct subpopulations of white adipocyte precursor cells (APCs) and adipocytes that display unique, cell-autonomous gene expression profiles. In addition to the large expansion of adipose tissue mass through the hypertrophy of adipocytes, numerous studies have found extensive adipocyte cell death occurs within visceral fat during obesity. Concomitantly, extensive macrophage infiltration occurs in adipose tissue, and over 90% of the infiltrating macrophages are arranged around these dead adipocytes, forming crown-like structures (CLS). Together, the death of adipocytes and the macrophage infiltration lead to lipotoxicity and chronic inflammation that are widely considered causative factors for systemic insulin resistance and diabetes. Several lines of preliminary evidence are presented in this proposal that indicate: 1) CLS are not homogenously distributed within adipose tissue, but occur in distinct clusters in the visceral adipose tissue, 2) subpopulations of adipocytes display a similar clustering, 3) adipocyte subpopulations have different susceptibility to TNF? induced apoptosis, 4) adipocyte subpopulations that are highly susceptible to TNF? reside preferentially in visceral fat, and 5) chronic high fat diet leads to a loss of these susceptible adipocyte subpopulations from visceral fat. Thus, the central hypothesis, unifying these findings, is that the clustering of CLS within a single fat pad are mediated, at least in part, by the differential response to inflammatory cytokines and susceptibility to apoptosis of adipocyte subpopulations. This hypothesis will be addressed in two specific aims. In the first aim, the effect of obesity on APCs and adipocytes subpopulations will be determined by placing mouse models which label these adipocyte subpopulations on a high fat diet. In the second aim, the differential response of APC and adipocyte subpopulations to TNF? will be determined by assessing 1) intracellular signaling, 2) insulin resistance, 3) adipogenesis, 4) lipolysis, and 5) apoptosis. These studies are expected decipher a unique cellular mechanism that controls the differential susceptibility of adipocytes to apoptosis that occurs during obesity. The proposed studies have great potential to fundamentally alter our current understanding of inflammation in adipose tissue, have broad implications in adipose tissue biology, and represent a first step in a continuum of research that is expected to lead to targeted interventions to combat the growing epidemic of type 2 diabetes.